GC/MSn analysis of the crude reaction mixtures from Friedel–Crafts acylation: Unambiguous identification and differentiation of 3‐aroylbenzofurans from their 4‐ and 6‐regioisomers

Rationale 3‐Aroylbenzofurans and their 2‐nitrophenyl derivatives constitute fundamental intermediates for the synthesis of target compounds with pharmaceutical properties. However, their preparation via the Friedel–Crafts acylation of 2‐phenylbenzofurans, using Lewis acid as catalyst, often leads to mixtures of regioisomeric aroylbenzofurans that can be challenging to distinguish, thus preventing the reaction characterization. Method We report a method for the unambiguous identification and differentiation of the desired 3‐benzoyl isomers from their 4‐ and 6‐regioisomers in a crude reaction mixture using gas chromatography coupled to multiple‐stage mass spectrometric (GC/MSn) analysis performed in collision‐induced dissociation (CID) mode. Results Upon electron ionization, each set of isomers displayed nearly identical mass spectra. MSn revealed fragmentation patterns that varied in the location of the benzoyl group on the benzofuran scaffold: CID experiments performed on the molecular ion allowed the distinction of the 3‐acyl isomers from the 4‐ and 6‐regioisomers; CID experiments on the [M − Ar]+ ion allowed the distinction of the 4‐benzoyl from the 6‐benzoyl regioisomer, when the nitro group is located on the 2‐phenyl ring. Moreover, the unusual loss of OH• radical allowed ascertaining the position of the nitro group in 3‐acyl regioisomers bearing the NO2 group. The origin of the diagnostic OH• loss was investigated through MSn experiments using 18O‐labelled 3‐benzoyl derivatives. Conclusions The method allows the rapid characterization of crude reaction mixtures of benzoylbenzofurans using solely GC/MSn analysis, simplifying the workflow of extensive isolation and purification for structure elucidation.


| INTRODUCTION
3-Aroylbenzofurans represent an important class of heterocyclic compounds in current pharmaceuticals use or development. [1][2][3][4] As a result, various methods for the synthesis of 3-aroylbenzofurans have been reported in the literature. The Friedel-Crafts acylation of aromatic compounds is one of the most widely used chemical reactions for the preparation of aromatic ketones at both academic and industrial levels. 5 However, during the Friedel-Crafts aroylation of 2-substituted benzofurans, various positions of the benzofuran ring are also acylated. 6 More specifically, although aroylation occurs predominantly at the 3-position, for most aroyl chlorides aroylation also occurs at the 6-position and to a lesser extent at the 4-position (Scheme 1; Figures 1A and 1B). 7 A different regioisomer distribution is observed in the acylation of benzofurans deactivated by a nitro group on the 2-phenyl ring ( Figure 1C). In this case, Friedel-Crafts acylation leads to a mixture of regioisomers where the expected 3-acyl isomer is formed as a minor product, reasonably because electron-withdrawing groups attenuate the reactivity and influence position selectivity. 8 Monitoring the reaction progress is of great interest in modern organic synthesis and drug research. However, thin-layer chromatography is often ineffective for the analysis of complex reaction mixture, such as that of various benzoylbenzofuran regioisomers. Moreover, the purification and isolation of each benzoyl isomer from their mixtures is often a very laborious process that requires many manual steps to generate sufficiently pure compounds for identification and reaction characterization. In a worst case, the minor component cannot be separated. 9 Hence, the need for sensitive methods that can rapidly identify benzoylbenzofuran regioisomers becomes crucial from this standpoint, especially for 3-benzoyl-2-phenylbenzofurans containing nitro groups, as they could provide convenient intermediates in the preparation of more complicated compounds. [10][11][12] In this paper, we report the gas chromatography coupled to multiple-stage mass spectrometric (GC/MS n ) analyses of three programmed from 80 C (held for 5 min) to 280 C at 20 C/min (held for 5 min); this was increased to 300 C at 20 C/min (held for 5 min). The temperature was then ramped to 350 C at 20 C/ min. The transfer line was maintained at 180 C and the injector port (30:1 split) at 290 C. MS n analyses were replicated five times.
The relative standard deviations of the relative peak intensities were below 15% (Tables S1-S18); in the measurements, very small peak are usually excluded and we used an intensity cutoff of 2%.

| Materials and reagents
All reagents and solvents were purchased from Sigma-Aldrich S.r.l. (Milan, Italy) and Carlo Erba Reagents S.r.l. (Milan, Italy) and used without further purification.
Reference compounds 1a, 1b and 1c were prepared as reported in our previous works. 14 Compounds 1d and 1e were obtained by the reaction of 2-(4-methoxybenzyloxy)benzyltriphenylphosphomium bromide and 4-nitrobenzoyl chlorides in the presence of Et 3 N in toluene. 15 All benzoylbenzofuran isomers and the labeled derivatives were isolated and characterized using 13 C NMR and 1 H NMR as described in the supporting information and in previous reports. 14-16 Isomers 2a, 2ad 5 , 2b and 2c were further purified using high-performance liquid chromatography.

| Separation and differentiation of isomers 1a-3a
The total ion current chromatogram of the reaction products from the Friedel-Crafts acylation of 2-phenylbenzofuran with benzoyl chloride is reported in Figure 1A. The three isomers so obtained are well separated, and the ion intensities of the isomers are different from each other. After purification, we found that acylation leads predominantly to the 3-acyl isomer 1a together with traces of the ( Figures 2E and 2H). Instead, 2a and 3a display the ion at m/z 221 as the base peak, which is particularly weak for the 3-benzoyl isomer 1a.
This ion corresponds for all isomers to the loss of radical C 6 H 5 • from the benzoyl group, as supported by the mass shift to m/z 226 in the spectra of the deuterium analogues 1ad 5 -3ad 5 (Figures S1 and S2). does the ion at m/z 165 constitute the base peak ( Figure 2C), whereas the ion at m/z 193 is the major peak for isomers 2a ( Figure 2F) and 3a ( Figure 2I).

| Separation and differentiation of isomers 1b-3b
The total ion current chromatogram of the Friedel-Crafts acylation of 2-phenylbenzofuran with 4-nitrobenzoyl chloride is shown in

| Separation and differentiation of isomers 1c-3c
The Friedel-Crafts acylation of 2-(4-nitrophenyl)benzofuran with benzoyl chloride leads to a mixture of regioisomers where the desired 3-acyl derivative is formed as a minor product ( Figure 1C). The EI mass spectra of the nitrobenzoyl isomers 1c-3c ( Figures 4A, 4E and 4H) show a rather similar mass fragmentation behavior, i.e. the formation of